We propose to continue our studies on human inborn errors of metabolism using cell culture systems. Our emphasis is on the genetic diseases that produce deficiencies of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex (i.e. maple syrup urine disease, MSUD), or the novel bifunctional aminoadipic semialdehyde synthase (i.e. familial hyperlysinemias, FH). Recent advances in understanding the molecular basis of inherited metabolic disorders have focused on relatively simple proteins. Little is known concerning the mutational processes or the coordination among multiple genetic loci of multienzyme complexes or bifunctional proteins. This information is needed to improve diagnosis and treatment of these patients. Our labortories have recently cloned the cDNA's of the E1 alpha and E2 subunit of the mammalian BCKAD complex. Using cultured cells as a model, our approach to the molecular basis of MSUD will be as follows: i) Protein and mRNA studies: Western and Northern blot analyses will be carried out with cultured normal and MSUD cells. Antibodies and cDNAs to each subunit (E1 alpha, E1 beta and E2) of the BCKAD complex will be used as probes. ii) DNA studies: Restriction-site polymorphisms in the E1 alpha and E2 loci among MSUD families will be explored to develop sensitive prenatal diagnosis at the DNA level. Nucleotide sequence in mutant mRNAs will also be determined to define mutations in MSUD. iii) Gene transfer experiments: Attempts will be made to transfer a full-length bovine E2 cDNA into E2- deficient MSUD cells via a shuttle or retroviral vector, and the expression of the transfected cDNA will be examined. Similar studies will be carried out with E1 subunits. iv) Gene isolation and characterization: The genes encoding the human E1 alpha and E1 beta will be isolated. Structures and polymorphic maps of these genes will be determined. v) Chromosomal localization: Genetic loci encoding E1 alpha, E1 beta and E2 subunits on human chromosomes will be identified. To study FH, our efforts will focus on protein chemistry and cloning of the bifunctional aminoadipic semialdehyde synthase discovered in our laboratories. Peptide sequencing, kinetic studies and sedimentation analysis of the native synthase and its isolated reductase and dehydrogenase domains will be carried out. Antibodies to the synthase and each domain will be produced. Full-length cDNA for the bovine and human synthase will be isolated from an expression library in lambda gtll or lambda ZAP. The antibody and cDNA probes will be used to define the molecular basis of FH types I and II. Approaches similar to those described for MSUD will be adapted for the FH studies.